NAD (P) H: (quinone acceptor) oxidoreductase (quinone reductase; QR) is a cytosolic, FAD-containing flavoprotein that plays a major role in the detoxication of quinones and their metabolic precursors. It is widely distributed in mammalian tissues where it promotes obligatory two-electron reductions of many quinones to hydroquinones. QR is induced by many xenobiotics, and the protective effects of many anticarcinogens may be attributed, at least in part, to their capacity to induce QR. Quinone reductase is also exquisitely sensitive to inhibition by dicoumarol and similar anticoagulants, and may play a critical role in the gamma- carboxylation of glutamate residues of blood coagulation factors. The mechanism by which this unusual flavoprotein carries out obligatory two- electron reductions is unclear. This project links the complementary expertise of two laboratories in x-ray crystal structure determination and the study of enzyme mechanisms by kinetic and spectroscopic methods with the goal of elucidating the structure and unique two-electron reduction mechanism of QR. In Aim 1, the structure of the rat liver QR-Cibacron Blue co-crystals will be determined and refined. In Aim 2, the binding of a number of flavin and nicotinamide nucleotides, Cibacron blue and other triazine dyes, and dicoumarol analogues will be examined by the aforementioned techniques. These ligands will then be incorporated into preformed rat QR crystals - or co-crystallized with the enzyme - and the structures of these QR complexes will be determined by difference Fourier methods. In Aim 3, the structural information obtained on the rat QR will be compared with the mouse liver QR, in order to obtain more generalized information on cofactor binding and flavoprotein catalysis of two-electron reductions. Knowledge of the structure and mechanism of QR is of central importance for understanding its protective functions and role in the biosynthesis of blood coagulation proteins.